Cyclosporin A and verapamil are substrates for P-glycoprotein. Both agents are known to reverse multidrug resistance in cells overexpressing P-glycoprotein. In this investigation, we have examined the effects of cyclosporin A and verapamil on multidrug resistance in HL60/AR cells that lack P-glycoprotein. In addition, a correlation was sought between an alteration in plasma membrane potential as measured with cationic dye DIOC5 and overexpression of P-glycoprotein. HL60/AR cells accumulated 3 fold less daunorubicin than HL60 cells. The drug accumulation defect and drug resistance in HL60/AR cells were partially corrected by verapamil and buthionine sulfoximine. However, cyclosporin A had no detectable effect on daunorubicin accumulation or drug resistance in HL60/AR cells. The multidrug resistant P338/ADR cell line overexpressed P-glycoprotein and exhibited depolarization of plasma membrane when compared to its corresponding drug sensitive parental cell line. In contrast, HL60/AR cells lacked P-glycoprotein and plasma membrane potentials were similar to those of drug sensitive HL60 cells. These results suggest that  verapamil modulates daunorubicin transport by a mechanism independent of P-glycoprotein,  the mechanisms of reversal of multidrug resistance by verapamil and cyclosporin A are distinct, and  the plasma membrane depolarization in multidrug resistant cell lines that overexpress P-glycoprotein, as determined by DIOC5, may be due to an increased efflux of cationic dye by P-glycoprotein, rather than a true measurement of plasma membrane potential in multidrug resistant cells.